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Bacteriophage T4 RNase H is a 5-to 3-nuclease that has exonuclease activity on RNA⅐DNA and DNA⅐DNA duplexes and can remove the pentamer RNA primers made by the T4 primase-helicase (Hollingsworth, H. C., and Nossal, N. G. (1991) J. Biol. Chem. 266, 1888 -1897; Hobbs, L. J., and Nossal, N. G. (1996) J. Bacteriol. 178, 6772-6777). Here we show that this exonuclease degrades duplex DNA nonprocessively, releasing a single oligonucleotide (nucleotides 1-4) with each interaction with the substrate. Degradation continues nonprocessively until the enzyme stops 8 -11 nucleotides from the 3-end of the substrate. T4 gene 32 single-stranded DNA-binding protein strongly stimulates the exonuclease activity of T4 RNase H, converting it into a processive nuclease that removes multiple short oligonucleotides with a combined length of 10 -50 nucleotides each time it binds to the duplex substrate. 32 protein must bind on singlestranded DNA behind T4 RNase H for processive degradation. T4 RNase H also has a flap endonuclease activity that cuts preferentially on either side of the junction between single-and double-stranded DNA in flap and fork DNA structures. In contrast to the exonuclease, the endonuclease is inhibited completely by 32 protein binding to the single strand of the flap substrate. These results suggest an important role for T4 32 protein in controlling T4 RNase H degradation of RNA primers and adjacent DNA during each lagging strand cycle.During the replication of duplex DNA, the synthesis of the discontinuous lagging strand fragments is initiated by short RNA primers that must subsequently be removed and replaced by DNA. In the bacteriophage T4 system, the pentamer RNA primers made by the T4 primase-helicase are removed by a phage-encoded enzyme with RNase H activity that has been called T4 RNase H (1). Our initial characterization of this nuclease showed that it also had 5Ј-to 3Ј-exonuclease activity on double-stranded (ds) 1 DNA, raising the possibility that some DNA adjacent to the RNA primers is removed from the lagging strand fragments. In this paper we show that interaction between T4 gene 32 protein and RNase H controls how much DNA is removed each time T4 RNase H binds to its substrate.T4 DNA replication is carried out by a multienzyme system in which T4 DNA polymerase (gene 43) is tethered to the template by the gene 45 clamp protein that has been loaded on the DNA by the gene 44/62 protein complex. The gene 32 single-stranded (ss) DNA-binding protein covers unwound DNA at the fork and increases the processivity of the lagging strand polymerase. The primase-helicase composed of the gene 61 primase and gene 41 helicase makes the RNA primers on the lagging strand and unwinds the duplex DNA ahead of the leading strand polymerase. The gene 59 helicase assembly protein facilitates the loading of the primase-helicase, especially on DNA covered with 32 protein. After removal of the primer and perhaps adjacent DNA by RNase H, lagging strand fragments are joined by DNA ligase (gene 30) (for review, see Ref. 2).The ge...

Bacteriophage T4 rnh encodes an RNase H that removes ribopentamer primers from nascent DNA chains during synthesis by the T4 multienzyme replication system in vitro (H. C. Hollingsworth and N. G. Nossal, J. Biol. Chem. 266:1888-1897, 1991). This paper demonstrates that either T4 RNase HI or Escherichia coli DNA polymerase I (Pol I) is essential for phage replication. Wild-type T4 phage production was not diminished by the polA12 mutation, which disrupts coordination between the polymerase and the 5-to-3 nuclease activities of E. coli DNA Pol I, or by an interruption in the gene for E. coli RNase HI. Deleting the C-terminal amino acids 118 to 305 from T4 RNase H reduced phage production to 47% of that of wild-type T4 on a wild-type E. coli host, 10% on an isogenic host defective in RNase H, and less than 0.1% on a polA12 host. The T4 rnh(⌬118-305) mutant synthesized DNA at about half the rate of wild-type T4 in the polA12 host. More than 50% of pulse-labelled mutant DNA was in short chains characteristic of Okazaki fragments. Phage production was restored in the nonpermissive host by providing the T4 rnh gene on a plasmid. Thus, T4 RNase H was sufficient to sustain the high rate of T4 DNA synthesis, but E. coli RNase HI and the 5-to-3 exonuclease of Pol I could substitute to some extent for the T4 enzyme. However, replication was less accurate in the absence of the T4 RNase H, as judged by the increased frequency of acriflavine-resistant mutations after infection of a wild-type host with the T4 rnh(⌬118-305) mutant.Bacteriophage T4 DNA replication is carried out by a multienzyme complex composed of phage-encoded proteins (reviewed in references 20 and 21). The lagging strand of the DNA duplex is synthesized discontinuously, with each fragment initiated by an RNA pentamer (pppApCpNpNpN) made by the primase-helicase encoded by T4 genes 61 and 41. These RNA primers must be removed and the gaps must be repaired before the Okazaki fragments are ligated together. The genes for most of the T4 DNA replication proteins were identified in experiments of conditionally lethal phage mutants with phenotypes of arrested, delayed, or no DNA synthesis (1). No genes encoding RNase H activities suitable for primer removal were found in these early studies.In an effort to understand how the processing of Okazaki fragments is controlled during T4 DNA replication, we purified an RNase H activity from T4-infected cells (9). We found that the size and N-terminal sequence of this protein match those of a previously identified open reading frame, designated orfA (5, 6), and showed by cloning that orfA encodes a protein with RNase H activity. In vitro this T4 RNase H degrades the RNA strand in RNA-DNA hybrids, has 5Ј-to-3Ј nuclease activity on double-stranded DNA, and is able to remove primers from DNA chains made by the purified T4 replication complex. It does not degrade single-stranded RNA or DNA. To determine whether this enzyme is important for primer processing in vivo, we have now constructed and characterized a T4 mutant in which a...

We have used metabolic radiolabeling with oligosaccharide precursors, coupled with subcellular fractionation, to examine the distribution o f several classes o f asparagine-linked oligosaccharides during early development. In Dictyosteliurn, we have observed endoglycosidase H (endo H)-sensitive structures with sizes corresponding to 10 (HexlO) and 11 ( H e x l l ) hexose residues on the chitobiose core. Only Hexl 1 was detected as the major structure on fucosylated endo H-resistant species. All Hexl 1 species cofractionated with plasma membrane and secreted glycoproteins, whereas Hexl 0 appeared to be confined to intracellular membrane and soluble glycoproteins. Sulfated species correlated with lysosomal and secreted fractions, and glucose residues were markedly depressed in H e x l l of secreted glycoproteins. Outer branch structural studies have revealed several components of the endo H-sensitive species. Using a-mannosidase and p-hexosaminidase as diagnostic tools, species elucidated thus far are: a structure with 10 mannoses, a structure with nine mannoses and an intersecting N-acetylglucosamine, structures with three glucoses and seven or eight mannoses and several larger species with multiple blocks to digestion.